UniProt and Mass Spectrometry-Based Proteomics—A 2-Way Working Relationship

•High-quality, comprehensive, free resource of protein sequence and function.•Reference sets of whole-proteome sequence, including human, made available.•Includes isoforms, postprocessed chains, PTMs, variants, and functional domains.•Imports and displays peptide and PTM identifications from public domain repositories.•Access via website, API or ftp, download data in multiple community data formats. The human proteome comprises of all of the proteins produced by the sequences translated from the human genome with additional modifications in both sequence and function caused by nonsynonymous variants and posttranslational modifications including cleavage of the initial transcript into smaller peptides and polypeptides. The UniProtKB database (www.uniprot.org) is the world’s leading high-quality, comprehensive and freely accessible resource of protein sequence and functional information and presents a summary of experimentally verified, or computationally predicted, functional information added by our expert biocuration team for each protein in the proteome. Researchers in the field of mass spectrometry–based proteomics both consume and add to the body of data available in UniProtKB, and this review highlights the information we provide to this community and the knowledge we in turn obtain from groups via deposition of large-scale datasets in public domain databases. The human proteome comprises of all of the proteins produced by the sequences translated from the human genome with additional modifications in both sequence and function caused by nonsynonymous variants and posttranslational modifications including cleavage of the initial transcript into smaller peptides and polypeptides. The UniProtKB database (www.uniprot.org) is the world’s leading high-quality, comprehensive and freely accessible resource of protein sequence and functional information and presents a summary of experimentally verified, or computationally predicted, functional information added by our expert biocuration team for each protein in the proteome. Researchers in the field of mass spectrometry–based proteomics both consume and add to the body of data available in UniProtKB, and this review highlights the information we provide to this community and the knowledge we in turn obtain from groups via deposition of large-scale datasets in public domain databases. Mass spectrometry (MS)-based proteomics researchers aim to isolate, identify, and functionally characterize the protein profile of a cell, tissue, or organism of interest. Scientists may be interested in understanding how this profile changes as cells develop, age, respond to changes in external environmental conditions, or how the application of a xenobiotic, such as a drug, changes cellular behavior at the molecular level. In order for researchers to be able to determine the protein complement of a biological sample, they need to be able to map peptide or protein sequences to a reference resource, which not only enables the required identifications but also links these to the relevant organism and provides details of protein nomenclature, function, and additional sequence features such as posttranslational modifications (PTMs) and functional domains. The UniProt data resource (1UniProt ConsortiumUniProt: the universal protein knowledgebase in 2023.Nucl. Acids Res. 2022; https://doi.org/10.1093/nar/gkac1052Crossref Scopus (713) Google Scholar) provides a complete compendium of all known protein sequence data linked to a summary of the experimentally verified, or computationally predicted, functional information about that protein and is the most widely used protein sequence database employed by the proteomics community. This review is aimed at enabling proteomics scientists to not only download the optimal set of sequences for use in their search engine(s) but also to subsequently access as much information on their identified proteins as possible. It also describes how MS data are used to inform and improve UniProt records and how we plan to extend that relationship moving forwards. Sequence database search engines identify peptides from tandem mass spectra via comparison to a reference protein sequence database (2Verheggen K. Raeder H. Berven F.S. Martens L. Barsnes H. Vaudel M. Anatomy and evolution of database search engines-a central component of mass spectrometry based proteomic workflows.Mass Spectrom. Rev. 2020; 39: 292-306Crossref PubMed Scopus (67) Google Scholar). The selection of which reference sequence database to use is therefore critical to the eventual output of the search engine algorithm and the results of the study. Database sequence coverage is of paramount importance, in that a search engine cannot identify peptides from proteins that are not present in the selected reference dataset. However, the protein sequence database should also enable the user to manage their search space, i.e., restrict the number of possible peptides against which a spectrum is searched, as too large a search space affects search engine speed, specificity, and sensitivity. Two of the most commonly used techniques to enable this are restricting the sequence selection to only one per gene and/or ignoring potential peptide modification by PTMs; however, both of these decisions require the sacrifice of information. The UniProt Knowledgebase (UniProtKB) (1UniProt ConsortiumUniProt: the universal protein knowledgebase in 2023.Nucl. Acids Res. 2022; https://doi.org/10.1093/nar/gkac1052Crossref Scopus (713) Google Scholar) is increasingly the database of choice for the MS community, and here we explain the reasons behind this, how UniProtKB has been structured to serve proteomics use cases, and how users can access additional data to enable a more complete analysis of their biological and biomedical datasets. UniProtKB consists of reviewed UniProtKB/Swiss-Prot entries to which data have been added by our expert biocuration team and unreviewed UniProtKB/TrEMBL entries that are annotated by automated systems. While, for a limited number of species including Human, Saccharomyces cerevisiae, and Escherichia coli, there is a single reviewed entry for every known protein coding gene, for most species complete proteome coverage can only be achieved through a combination of reviewed and unreviewed entries. Reviewed entries contain the sequences of all the protein products encoded by one gene, each with a unique identifier. This includes protein isoforms expressed by that gene along with any shorter protein chain/peptide generated from the parent transcript. Isoforms are defined as sequences that can be generated from the same gene by a single or combination of biological events (alternative promoter usage, alternative splicing, alternative initiation, and ribosomal frameshifting). Isoforms yet to be manually integrated are maintained in unreviewed UniProtKB/TrEMBL entries but can be viewed in the corresponding reviewed entry on the website as a result of automated gene-centric mappings. To create a UniProtKB/Swiss-Prot entry, UniProt curators firstly examine the protein sequence and correct, where necessary, for errors caused by sequence annotation issues such as erroneously selected initiation sites or frameshifts. They identify peptide chains generated by posttranslational cleavage of the parent transcript. These chains may be associated with protein location and activation and are generated by the removal of a signal or transit peptide or be bioactive peptides with defined biological activities. Positional features described within the UniProt entry, such as binding sites, PTMs, and amino acid variants, are mapped to a selected representative (canonical) sequence which is the entry displayed by default on the website and in most download formats. All sequences (canonical and isoforms) are distributed in FASTA format as described in the ‘Accessing data’ section. The main source of protein sequences in UniProt is the translation of nucleic acid sequence submissions to the International Nucleotide Sequence Database Collaboration (INSDC) source databases (3Arita M. Karsch-Mizrachi I. Cochrane G. The international nucleotide sequence database collaboration.Nucl. Acids Res. 2021; 49: D121-D124Crossref PubMed Scopus (99) Google Scholar). Increasingly, these consist of the protein content of completely sequenced genomes, i.e., a proteome and are supplemented by proteomes sequenced and/or annotated by groups such as Ensembl (4Howe K.L. Achuthan P. Allen J. Allen J. Alvarez-Jarreta J. Amode M.R. Acids Res. 2021; 49: PubMed Scopus Google the for K. J. of the for in 2023.Nucl. Acids Res. 2022; Scopus Google for of human and 2022; PubMed Scopus Google Scholar) and K.L. M. P. 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